Lipid modulation of early G protein-coupled receptor signalling events

Abstract

Upon binding of extracellular ligands, G protein coupled-receptors (GPCRs) initiate signalling cascades by activating heterotrimeric G proteins through direct interactions with the α subunit. While the lipid dependence of ligand binding has previously been studied for one class A GPCR, the neurotensin receptor 1 (NTS1), the role the lipid environment plays in the interaction of activated GPCRs with G proteins is less well understood. It is therefore of interest to understand the balance of lipid interactions required to support both ligand binding and G protein activation, not least since some receptors have multiple locations, and may experience different membrane environments when signalling in the plasma membrane or during endocytosis. Here, using the sensitive biophysical technique of microscale thermophoresis in conjunction with nanodisc lipid bilayer reconstitution, we show that in more native lipid environments rich in phosphatidyl ethanolamine (PE), the Gαi1 subunit has a ~4-fold higher affinity for NTS1 than in the absence of native lipids. The G protein-receptor affinity was further shown to be dependent on the ligand-binding state of the receptor, with potential indication of biased signalling for the known antagonist SR142948A. Gαi1 also showed preferential interaction with empty nanodiscs of native lipid mixtures rich in PE by around 2- to 4-fold over phosphatidyl choline (PC)/phosphatidyl glycerol (PG) lipid mixtures. The lipid environment may therefore play a role in creating favourable micro-environments for efficient GPCR signalling. Our approach combining nanodiscs with microscale thermophoresis will be useful in future studies to elucidate further the complexity of the GPCR interactome.

Interaction of lipids with the neurotensin receptor 1

“…Recently, we showed that binding of neurotensin to NTS1, receptor homodimerisation, and coupling to G αi1 are greatly affected by the specific lipid composition of the bilayer [40,41], and Grisshammer and co-workers showed that the same holds for G q protein activation [12]. Here, we now analyse how major lipid species of the mammalian plasma membrane interact with the transmembranous region of rat NTS1 using a fusion protein termed NTS1-B.…”

Recent advances in nanodisc technology for membrane protein studies (2012–2017)

“…Dijkmam et al . demonstrated that the class A GPCR neurotensin receptor 1, when incorporated into nanodiscs rich in phosphatidylethanolamine, experienced the Gα i1 subunit having a fourfold higher affinity for the GPCR. While various other GPCRs have been incorporated into nanodiscs to study their function (Table ), the effect of various lipid compositions on GPCR functionality has not been thoroughly investigated.…”

Design: An assay based on single-polypeptide-chain heterodimeric A2AR/D2R and non-oligomerized fusions forin vivoanalysis of their allosteric receptor-receptor interactions

“…Sternweis and Robishaw reported thehigh affinity binding of a non-hydrolyzable GTP analog, GTPS, for G-protein in bovine brain membranes, with 0.37 nmol/mg of protein[73,74]. "Kinetic data on G protein activation are difficult to obtain,"[69], however, and parameters on the affinity between a GPCR and a G-protein have not clearly been reported so far, especially in cells[69], except for rhodopsin with Kd of nM ranges determined by cell-free, surface plasmon-waveguide resonance spectroscopy[75,76] and for a class A GPCR with Kd of around 100 nM by microscale thermophoresis[77]. A recent study, by double electron-electron resonance spectroscopy through paramagnetic labels carrying unpaired electrons that can be introduced commonly as nitroxides via usually sulfhydryl group of cysteine residues of proteins of interest[78], indicates "a minority population exhibiting substantial domain separation" between the GTPase and helical domains of G-protein [79], although "no crystal structure of a nucleotide-bound G protein has captured a domain-separated conformation-perhaps because such conformations are less populated and less amenable to crystallization than one with the domains in tight contact-"[80].…”